Adenomatous polyposis coli associates with the microtubule-destabilizing protein XMCAK

During cell division, the proper formation of a bipolar spindle and its function to segregate chromosomes requires precise coordination of microtubule-stabilizing and destabilizing activities. Globally destabilized, dynamic microtubules radiating from duplicated centrosomes are locally regulated by chromosomes. Proteins at the kinetochore of each sister chromatid mediate a dynamic attachment, allowing chromosome movement coupled to microtubule polymerization/depolymerization and error-correction mechanisms for improperly attached chromosomes. The tumor suppressor protein adenomatous polyposis coli (APC) stabilizes microtubules both in vitro and in vivo and is implicated in mitosis, although its mechanisms of action are not well characterized. Here, we show that in mitotic Xenopus egg extracts, the carboxyl-terminus of APC can associate with the amino terminus of the microtubule-destabilizing KinI, Xenopus mitotic centromere-associated kinesin (XMCAK), in a cytoplasmic complex. We find that like XMCAK, APC can localize to the centromere as well as the kinetochore region of mitotic chromosomes and does not require microtubules for chromosomal targeting in Xenopus egg extracts. We propose that the presence of these proteins in a complex brings together both positive and negative microtubule effectors, whose opposing activities may be regulated by additional factors, thereby providing precise control of both global and local microtubule dynamics.     

Adenomatous polyposis coli regulates axon arborization and cytoskeleton organization via its N-terminus

Conditional deletion of APC leads to marked disruption of cortical development and to excessive axonal branching of cortical neurons. However, little is known about the cell biological basis of this neuronal morphological regulation. Here we show that APC deficient cortical neuronal growth cones exhibit marked disruption of both microtubule and actin cytoskeleton. Functional analysis of the different APC domains revealed that axonal branches do not result from stabilized β-catenin, and that the C-terminus of APC containing microtubule regulatory domains only partially rescues the branching phenotype. Surprisingly, the N-terminus of APC containing the oligomerization domain and the armadillo repeats completely rescues the branching and cytoskeletal abnormalities. Our data indicate that APC is required for appropriate axon morphological development and that the N-terminus of APC is important for regulation of the neuronal cytoskeleton.     

Adenomatous polyposis coli proteins and cell adhesion

Adenomatous polyposis coli (APC) is an important tumour suppressor in the mammalian intestinal epithelium. It binds to beta-catenin and its role as a tumour suppressor depends predominantly on its ability to downregulate soluble beta-catenin, a key effector of the Wnt signalling pathway. However, epithelial cells have a distinct subcellular pool of beta-catenin, or Drosophila Armadillo, which functions as a structural component of adherens junctions. Notably, APC proteins can be associated with these adherens junctions, and recent evidence points to a role for APC in cellular adhesion. Thus, APC--like beta-catenin/Armadillo--may have a dual role in Wnt signal transduction and in cellular adhesion, which could be relevant to its activity as a tumour suppressor.     

A role of the mitotic spindle checkpoint in the cellular response to DNA replication stress

Replication stress is a frequent and early event during tumorigenesis. Whereas the cellular responses to a persistent block of replication fork progression have been extensively studied, relatively little is known about how cells respond to low-intensity replication stress. However, transient replication fork perturbations are likely to occur even more frequently in tumor cells than a permanent replication arrest. We report here that transient, low intensity replication stress leads to a rapid activation of the DNA replication checkpoint but to a significantly delayed apoptotic response in a small but significant number of cells. This late apoptotic response was independent of p53 and we found evidence for cell death during mitosis in a proportion of cells. To further explore the role of p53 in the response to replication stress, we analyzed mouse embryonic fibroblasts (MEFs) deficient of p53 in comparison to wild-type or p63- or p73-deficient MEFs. We detected a significant increase of apoptosis and morphological signs of failed mitosis such as multinucleation in p53-deficient MEFs following replication stress, but not in wild-type or p63- or p73-deficient cells. Multinucleated p53-deficient MEFs frequently retained cyclin B1 expression indicating a persistently activated mitotic spindle checkpoint. Collectively, our results suggest that the cellular response to replication stress involves the mitotic spindle checkpoint in a proportion of cells. These findings imply that the mitotic spindle checkpoint may act in concert with DNA damage and cell-cycle checkpoints as an early anti-tumor barrier and provide a possible explanation for its frequent relaxation in human cancer.     

Death-associated protein 3 regulates cellular senescence through oxidative stress response

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.     

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal “basic” domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly–promoting factors with complementary activities.     

Adenomatous polyposis coli (APC) differentially regulates beta-catenin phosphorylation and ubiquitination in colon cancer cells

Most colorectal cancers have mutations of the adenomatous polyposis coli (APC) gene or the beta-catenin gene that stabilize beta-catenin and activate beta-catenin target genes, leading ultimately to cancer. The molecular mechanisms of APC function in beta-catenin degradation are not completely known. APC binds beta-catenin and is involved in the Axin complex, suggesting that APC regulates beta-catenin phosphorylation. Some evidence also suggests that APC regulates beta-catenin nuclear export. Here, we examine the effects of APC mutations on beta-catenin phosphorylation, ubiquitination, and degradation in the colon cancer cell lines SW480, DLD-1, and HT29, each of which contains a different APC truncation. Although the current models suggest that beta-catenin phosphorylation should be inhibited by APC mutations, we detected significant beta-catenin phosphorylation in these cells. However, beta-catenin ubiquitination and degradation were inhibited in SW480 but not in DLD-1 and HT29 cells. The ubiquitination ofbeta-catenin in SW480 cells can be rescued by exogenous expression of APC. The APC domains required for beta-catenin ubiquitination were analyzed. Our results suggest that APC regulates beta-catenin phosphorylation and ubiquitination by distinct domains and by separate molecular mechanisms.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

Ca regulates the subcellular localization of adenomatous polyposis coli tumor suppressor protein

Microtubule (MT) plus-end tracking proteins (+TIPs) are involved in the regulation of MT plus-end dynamics and stabilization. It was reported previously that an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by disruption of the plasma membrane stimulates rearrangement of MTs [T. Togo, Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site, J. Cell Sci. 119 (2006) 2780-2786], suggesting that some +TIPs are regulated by Ca²⁺. In the present study, the behavior of adenomatous polyposis coli (APC) following an increase in [Ca²⁺]_i was observed using Xenopus A6 epithelial cell expressing GFP-tagged APC. An increase in [Ca²⁺]_i by cell membrane disruption or by ionomycin treatment induced dissociation of APC without depolymerizing MTs. Inhibition of a tyrosine kinase and GSK-3β suppressed APC dissociation upon an increase in [Ca²⁺]_i. Western blotting analysis showed that Ca²⁺ transients activated GSK-3β through a tyrosine kinase. These results suggest that Ca²⁺ stimulates redistribution of APC through a tyrosine kinase- and GSK-3β-dependent pathway.     

Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response.

Many of the proteins that operate at the replication fork in Escherichia coli have been defined genetically. These include some of the subunits of the DNA polymerase III holoenzyme, the DnaB replication fork helicase, and the DnaG primase. The multiprotein primosome (which includes the DnaB and DnaG proteins), defined biochemically on the basis of its requirement during bacteriophage phi X174 complementary-strand synthesis, could serve as the helicase-primase replication machine on the lagging-strand template. In order to determine if this is the case, we have begun an investigation of the phenotypes of mutants with mutations priA, priB, and priC, which encode the primosomal proteins factor Y (protein n'), n, and n", respectively. Inactivation of priA by insertional mutagenesis resulted in the induction of the SOS response, as evinced by induction of a resident lambda prophage, extreme filamentation, and derepression of an indicator operon in which beta-galactosidase production was controlled by the dinD1 promoter. In addition, the copy numbers of resident pBR322 plasmids were reduced four- to fivefold in these strains, and production of phi X174 phage was delayed considerably. These results are discussed in the context of existing models for SOS induction and possible roles for the PriA protein at the replication fork in vivo.     

Phosphatase PHLPP2 regulates the cellular response to metabolic stress through AMPK

PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.Subject terms: Cancer metabolism, Stress signalling     

The F-box protein Dia2 regulates DNA replication

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage in both S and G2/M phases of the cell cycle. These defects are likely a result of the absence of SCF(Dia2) activity, as a Dia2 DeltaF-box mutant shows similar phenotypes. Interestingly, prolonging G1-phase in dia2 cells prevents the accumulation of DNA damage in S-phase. We propose that Dia2 is an origin-binding protein that plays a role in regulating DNA replication.     

Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli

The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate. The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1–2 min and restore a higher initiation frequency. High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process. It is inferred that turgor could control DNA replication and cell division in two separate ways. Glycine betaine action is not mediated by K⁺ ions as the internal level of K⁺ ions is not modified significantly following glycine betaine accumulation.     

ATR: a master conductor of cellular responses to DNA replication stress

The integrity of the genome is constantly challenged by intrinsic and extrinsic genotoxic stresses that damage DNA. The cellular responses to DNA damage are orchestrated by DNA damage signaling pathways, also known as DNA damage checkpoints. These signaling pathways play crucial roles in detecting DNA damage, regulating DNA repair and coordinating DNA repair with other cellular processes. In vertebrates, the ATM- and Rad3-related (ATR) kinase plays a key role in the response to a broad spectrum of DNA damage and DNA replication stress. Here, we will discuss the recent findings on how ATR is activated by DNA damage and how it protects the genome against interference with DNA replication.